We are using high-pressure freezing and freeze substitution fixation, followed by serial thin sectioning to examine the mitotic spindle of Saccharomyces cerevisiae. This organism has a particularly rich tradition of cell cycle genetics, and many temperature-sensitive-for-growth mutants are available. We have modeled mitotic spindles of wild-type cells to determine the number and organization of its microtubules. A paper describing our results has been published (Winey et al., 1995) We are now modeling the defective mitotic spindles found in mutant cells. This has begun with an analysis of the cdc20 mutant in collaboration with Dr. Daniel Burke (University of Virginia). Based on immunofluorescent staining of microtubules, cdc20 mutants arrest with aberrant spindles. Our preliminary analysis of cdc20 cells reveals clear abnormalities in microtubule numbers and organization. We are also collaborating with Dr. Tim Huffaker (Cornell University) to model spindles in various tubulin mutants. We suppose that some mutant alleles may have phenotypes detectable only by high resolution analysis of the spindle. The second project objective is the high resolution localization of spindle components by immuno-EM localization. The position of colloidal gold particles in spindle models may lead to sub-spindle localization of some spindle components. We have been collaborating with Dr. Mark Rose (Princeton) and Dr. David Roof (University of Pennsylvania) to localize the spindle kinesin-like protein encoded by the KIP1 gene. Initial attempts to localize the KIP1 protein using an epitope-tagged version of the gene have failed, but we are currently awaiting a poly-clonal antibody to this protein. It is our belief that a high resolution model of the S. cerevisiae spindle, a catalog of mutant phenotypes and ultra-structural localization of spindle components will help to elucidate the action of this complex cellular apparatus.